External morphometric and microscopic analysis of the reproductive system in in- vitro reared stingless bee queens, Heterotrigona itama, and their mating frequency

Stingless bees, prevalent in tropical and subtropical regions, are a tribe of eusocial bees that are crucial pollinators for economic crops and native plants and, produce honey and pollen. However, colony expansion is limited by a shortage of queens for new colonies. Therefore, mass artificial rearing of virgin queens could address this in commercially managed meliponiculture. Furthermore, the in vitro rearing of queen stingless bees can improve meliponiculture management and conservation efforts. Herein, we explored the efficacy of in vitro queen rearing for Heterotrigona itama, assessing the queen’s body size, reproductive organ size (ovary and spermatheca), acceptance rate into new, small colonies, and mating frequency. H. itama larvae developed into queens when fed with 120 μL–150 μL of larval food, resulting in in vitro queens having body sizes similar to those of naturally produced queens. Microscopic analysis revealed well-developed ovaries and spermathecae in in vitro-reared queens, unlike the smaller ovaries and the absence of spermathecae in the naturally produced workers. Acceptance of in vitro-reared queens was independent of worker age, and mating frequency was low but not significantly different from naturally produced queens. These findings could enhance stingless beekeeping practices and conservation efforts for the native stingless bee species.

Meliponiculture (stingless beekeeping) has seen significant growth in Thailand, especially in rural villages, where it is promoted as a secondary profession.The rich diversity of stingless bee species has fueled this revival.However, a primary challenge Thai stingless beekeepers face is the difficulty of colony splitting owing to a scarcity of virgin queens.Successful propagation requires precise timing, considering food availability and drone presence, and identifying healthy brood combs [10].Therefore, this scarcity of virgin queens for establishing new colonies may markedly compromise management strategies for propagating stingless bee colonies [11].
Unlike honeybees of the genus Apis, stingless bees do not progressively feed larval food to their brood.Instead, they practice mass provisioning, using prepared larval food, a mixture of fermented pollen, honey, and glandular secretions of nurse bees, rich in associated with microbial communities [12].This mixture is placed in a brood cell, and the queen lays an egg on top of it.Workers subsequently close the open brood cells until adult stingless bees emerge [13,14].Most Meliponine species rear young queens in the largest brood cells, called royal cells, whereas workers and males are reared in smaller ones [15,16].Royal brood cells can contain up to eight times more larval food than for worker broods in some stingless bee species [15].Consequently, female larvae develop into queens when they receive the most larval food [16,17].
Three techniques, namely splitting, bridging, and splitting-bridging, have been employed for artificial propagation in Heterotrigona itama [18].While bridging and splitting-bridging methods yield high-quality hive compositions, the presence of the original queen often prevents the development of queen brood cells.Artificial queen-rearing techniques for stingless bees involve overfeeding 1-3-day-old female larvae in vitro [16,17,19], as caste determination is primarily based on the amount of larval food [20].Female larvae destined to become queens receive more food in larger royal cells, whereas those becoming workers are reared in smaller brood cells [20].In vitro rearing of virgin queens can address the low natural production of virgin queens in stingless bee species, leading to a rapid increase in new colonies [11,21,22] and spitting success rate.
At least 33 stingless bee species have been reported in Thailand [23][24][25].Among them, H. itama (Cockerell, 1918) is particularly effective in artificial wooden hive boxes.Beekeepers propagate H. itama colonies for the sale and production of honey, and rent them for the pollination of crops.These colonies can be sold for 3000-5000 THB (85-140 USD) each (AR: personal survey).Additionally, Thai H. itama honey sells for 1200-1500 THB (35-45 USD) per kg, which is approximately 10 times the price of honey from Thai Apis mellifera and approximately three times that of honey from native Thai Apis species (A.florea, A. dorsata, and A. cerana) [3].
Hence, in this study, we examined the impact of larval food quantity on the external morphology and reproductive organs of in vitro-reared H. itama queens.Furthermore, we determined the acceptance rate of these queens into small artificial queenless colonies.We compared the mating frequency between in vitro-reared and naturally raised H. itama queens in commercial apiary conditions, suggesting that in vitro rearing H. itama queens are efficient for rapidly increasing colonies.This approach improved meliponiculture management in Thailand and could help prevent overhunting of wild stingless bee colonies.

Stingless bee and study sites
The experiments were conducted at the Department of Entomology, Kasetsart University, Bangkok, Thailand.Furthermore, the amount of larval food and acceptance of in vitro-reared queen into new colonies were determined at two commercial apiaries located in Yi-ngo district, Narathiwat province (06˚23 0 31 00 N; 101˚41 0 46 00 E) and Chulabhorn district, Nakhon Si Thammarat province (08˚01 0 06 00 N; 99˚48 0 29 00 E), southern Thailand.The stingless bee species used was H. itama (Cockerell, 1918).This species is a medium-sized stingless bee, with a worker body length of approximately 5.5−5.6 mm.Perennial colonies comprise of approximately 8,000-10,000 worker bees and a single physogastric queen.

Ethics statement
Stingless beekeepers were prepared to employ this commercially significant species in this study.Written consent was offered for their approval following the stated purpose and methods outlined, and they agreed to carry out the study on their farms.Farm owners then participated in colony quality evaluation and facilitated experimental arena preparation.Additionally, no endangered or protected species were included in the field survey.The sample number collected was kept to a minimum, and ethical treatment was applied according to the research standards.The Animal Experiment Committee of Kasetsart University, Thailand (approval no.ACKU66−AGR−015) approved all the animal-based experiments.

Amount of larval food
(a) Procedure to determine the amount of larval food.We assessed and compared the larval food amount in the queen brood cells (n = 82 brood cells; 5.12 ±1.82 brood cells/colony, ranging from 2-10 brood cells/colony) to that in the worker brood cells (n = 290 brood cells; 18.12 ±2.96 brood cells/colony, range 12-20 brood cells/colony) of H. itama.These brood cells were collected from 16 unrelated parental colonies of two commercial apiaries in Narathiwat and Nakhon Si Thammarat provinces.Ideal conditions for the colonies include numerous individual workers, strong single queens, and the absence of diseases and parasites.
Brood combs were collected in a manner that did not harm the colonies.The recently built queen and worker brood cells were gently removed from the hive box using a knife and scalpel to avoid massive colony destruction.Subsequently, the brood cells were kept in plastic containers (15 × 20 × 7 cm) in the dark to minimize potential microorganism exposure.The queen cells were separated from the edges of the brood combs to prevent excessive cell damage.Calibrated microcapillary tubes with an auto-micropipette setup [17], with slight modifications, were used to meticulously collect and determine the larval feeding amounts from individual recently capped-brood cells, each containing a single egg [19].Typically, newly constructed worker brood cells are dark brown, and as they progress into larvae, they lighten in color since the worker bees remove the cerumen [10].
(b) Comparing the larval food amounts.We employed a one-sample Kolmogorov-Smirnov test to evaluate data normality and compare the larval food amounts between the queen and worker brood cells.After discovering that the data were non-parametric (Kolmogorov-Smirnov; Z = 8.627, P<0.001), we used a chi-square test to compare the two groups (queens vs. workers).We first evaluated data normality using the one-sample Kolmogorov-Smirnov test to compare the larval food amount in the queen cells between the two meliponaries.As these data were also non-parametric (Kolmogorov-Smirnov; Z = 1.739,P = 0.0047), we used the Mann-Whitney U test to compare between meliponaries.All data analyses were performed using IBM SPSS Statistics 22 [26].

Procedure involving in vitro queen rearing
(a) Harvesting larval food.This procedure aimed to collect adequate larval food for in vitro queen rearing.As mentioned earlier, recently capped worker broods were removed from the hive boxes.In H. itama, the brood cells are arranged side by side in multiple horizontal layers [3].Only one or two layers of new worker brood cells were taken from each cell to minimize colony disruption.Brood cells were subsequently opened using fine-tipped forceps.Before collecting the larval food, the eggs were removed using a sterile needle.Brood combs without eggs were transferred into a 50-ml centrifuge tube and subsequently squeezed to extract the larval food liquid.In this study, we compared two larval food levels, 120 and 150 μL, to determine the quantity of the larval food required to produce queens equal in size to those naturally produced.This value was based on the results of the present experiment, which determined the larval food amount in the queen brood cells (see results) and the findings of Razali et al. [10].We added approximately 20% (totaling 150 μL) of the same larval food amount to the normal queen cells [16] to ensure sufficient food for larval queen development.
(b) Queen rearing procedure.In the laboratory, 120 μL (three plates; n = 288 wells) and 150 μL (three plates; n = 288 wells) of larval food was transferred into each well of the 96-well ELISA plates, providing sufficient space for each H. itama larva.First, the larval food was homogenized using a manual pipette (drawing up larval food and dispensing it several times) and was provided to the artificial cells.The new brood cells containing eggs were carefully opened, and the eggs were subsequently carefully removed from the brood cells using a sterilized Chinese queen grafting tool (Fig 1A) and transferred to each well (Fig 1B).To retain their original orientation, all eggs were vertically placed on the larval food.

Microscopic analysis of the reproductive system
We conducted a comparative analysis of the ovaries of in vitro queens and newly emerged workers obtained from the H. itama natural colonies to explore virgin queen production under in vitro conditions.Workers were recruited randomly from the apiaries for this purpose.Fresh samples of eleven in vitro queens and twenty newly emerged workers (five from each of four colonies) were placed in Petri dishes.Subsequently, these samples were fixed in a phosphate-buffered saline (136.9Mm NaCl, 8.1 Mm Na2HPO4, 2.7 Mm KH2PO4) with a pH of 7.1 to prevent the reproductive organs from drying out during the dissection process, following the protocol proposed by Razali and Razak [10].For precision, the dissection was performed using super-fine-tip forceps under an Olympus SZ51 stereomicroscope.Immediately following the dissection, images of the reproductive system were captured using a Leica EZ4W microscope.

Morphometric analysis
(a) Obtained natural virgin queen of H. itama.Thirty capped queen cells were collected from five colonies in two apiaries in Nakhon Si Thammarat (n = 2) and Narathiwat (n = 3) provinces.Capped queen cells were kept separate from each other.Next, each queen cell was placed in an artificial cage and reintroduced into the colony from where it originated.Adult virgin queens in the queen cages were collected upon emergence and preserved in 70% ethanol for morphometric analysis.
(b) Morphometry measurement.All in vitro queens (n = 11), natural virgin queens (n = 22), and young adult workers (n = 30) were dissected using fine-tipped forceps under a stereomicroscope.This method included the antenna, mandible, head (with compound eyes), right forewing, right hindwing, right hindleg (with the femur, tibia, and basitarsus), 4 th and 5 th tergites, and 4 th and 5 th sternites.All body parts were mounted onto microscope slides and photographed using a digital camera attached to a stereomicroscope (Leica EZ4 W/E; Leica).Leica LAS EZ software (Leica, Wetzlar, Germany) measured all morphometric traits according to previously published methods [27][28][29][30].Herein, 34 morphometric characteristics of the bee samples were measured and analyzed (S1 Fig) .(c) Morphometry analysis.Means and standard errors of all characteristics were calculated.The G-test, included in IBM SPSS Statistics 22, was used to examine the skewness of the character sizes [26].Discriminant analysis was used to differentiate in external characteristics among the in vitro virgin queens, natural virgin queens, and young adult workers.Samples were grouped into three categories to compare different sizes: in vitro virgin queens, natural virgin queens, and young adult workers.The characters' centroid size (CS) was calculated to measure the overall size of the stingless bee specimens [31] and was used to assess whether bees from various groups differed in size.The CS of all characters among the three H. itama groups was compared using discriminant analysis implemented in IBM SPSS Statistics 22 [26].

In vitro queen acceptance
(a) Introducing the in vitro queen into the new colony.We investigated the acceptance of queens reared in vitro into new artificial queenless colonies.First, we produced two sets of artificial small queenless colonies (n = 27) by introducing approximately 500 workers of various ages (artificial queenless colonies a (AQCa): newly emergent workers only; artificial queenless colonies b (AQCb): newly emergent and old workers) collected from strong queenright colonies.Second, we introduced two brood combs containing approximately 200 larvae close to emergence.Finally, approximately 100 g of pollen and 200 g of honey pots were placed in the corners of the hive box.After 2-3 day, approximately 1-week-old queens reared in vitro were introduced into queen cages (S1 Video) and subsequently placed inside artificial queenless colonies (AQCa = 14 colonies and AQCb = 13 colonies).Their behavior toward the workers was recorded.If the in vitro queens remained alive for at least 7 days after introduction, they were declared as being accepted.Additionally, we deemed the in vitro rearing of the queens as successful when the vibrated their wings, were not attacked by workers, and exhibited trophallaxis with other bees [17].All experimental procedures were conducted at the Nakhon Si Thammarat apiary.
(b) Analyzing the acceptance of in vitro queens.We evaluated the likelihood of the successful in vitro adoption of the queens into artificial queenless colonies using an odds ratio test with a 95% confidence interval (CI).The acceptance rate of the in vitro queens was compared between two types of artificial queenless colonies containing (a) only non-pigmented workers and (b) non-pigmented and old workers using the Pearson's chi-square test (n = 9999; Monte Carlo simulation).All data analyses were performed using IBM SPSS Statistics 22 [26].

Analyzing the mating frequency of the in vitro queen
(a) Worker sample collection.Ten young adult workers were collected from 3-monthold colonies containing in vitro queens.They were preserved in 95% (v/v) ethanol to evaluate the number of fathering genotypes of in vitro queen-headed colonies.All young adult worker samples were directly collected from the brood combs inside each colony.The samples were stored at −20˚C until further use for DNA extraction.Eight in vitro queen-heading colonies were used in this study.
(b) DNA extraction, polymerase chain reaction (PCR) condition, and genotyping.Genomic DNA was extracted from the right hind leg of an individual worker bee using a 5% (w/v) Chelex solution (Chelex1100; BIO-RAD), following the process described by Walsh et al. [32] with slight modifications.Three microsatellite target sequences were amplified via PCR using fluorescent-labeled primers.The microsatellite loci used were TC4.287, TC7.13, and TC3.155 [33].S1 Table lists the primer details.The PCRs were conducted using a T100™ thermal cycler (BIO-RAD) containing 1× Multiplex PCR Master Mix (Green HotStart PCR Master Mix, Biotechrabbit), 1 μL of each primer (20 μmol/L), and 1 μL of genomic DNA template, and distilled water up to a total volume of 10 μL.All microsatellite loci were amplified using a standard PCR program of 94˚C for 5 min, followed by 35 cycles of 94˚C, 56˚C, and 72˚C for 30 s each, and finally 72˚C for 10 min.Samples without DNA were included in all the plates as negative controls.The PCR products were subsequently sent to Macrogen (Seoul, South Korea) for fragment analysis.The resulting data files were analyzed for allele size determination using GENEMAPPER (Applied Biosystems).
(d) Analyzing the mating frequency.The effective mating frequency (me) within each colony, with correction for a finite sample size, was calculated according to the method followed by Tarpy and Nielsen [38].To determine intracolonial relatedness, the average relatedness, r, weighted according to the relative proportions of each subfamily and corrected for finite sample size, was calculated for each colony according to Oldroyd and Moran [39].Furthermore, the observed paternity frequency (k) was calculated for a common number of workers scored according to the method followed by Franck et al. [40] to make a valid comparison of the mating frequency between natural and in vitro-reared queens.

Amount of larval food in the queen and worker brood cells
This study aimed to determine the larval food amount required for the growth of H. itama queens and their workers under natural colony conditions.The larval food amount deposited in queen brood cells (119.53 ± 2.42 μL) was significantly higher than those found in the worker brood cells (18.22 ± 1.67 μL) in the normal queen-right colonies (χ 2 = 434.77,df = 90, P < 0.001) (Fig 2).No significant difference was found in the larval food amount deposited in the queen brood cells between apiaries (Mann-Whitney U test: U = 823.5,P = 0.878) and among colonies (Kruskal-Wallis Test: χ 2 = 19.43,df = 15, P = 0.195).Notably, the larval food deposited in the queen brood cells was approximately seven times higher than that in the worker brood cells under natural conditions.

In vitro larval development
Depending on the larval developmental stage, the average temperature and humidity in an incubator filled with distilled water and NaCl were maintained (Table 1).The eggs in this experiment took approximately 4−5 days to develop into larval forms.After hatching, the larvae began feeding on the food provided on the acrylic plates.Subsequently, the larvae turned into pupae after approximately 17 days (Table 1 and Fig 3).Larval queens reared in vitro developed for approximately 54 days (n = 90, 54.12 ±1.41) before emerging as adults in this study.

Worker and queen emergence in response to the amount of larval food
This study yielded 173 (60.07 ± 5.92%) and 186 (64.58 ± 3.76%) in vitro H. itama queens from 120 μL and 150 μL of larval food, respectively.No significant difference was found between the two larval food levels provided in response to queen emergence (t = 1.115, df = 4, P = 0.327).The eggs treated with the highest larval food amount (150 μL) resulted in 64.58 ± 3.76% queen and 6.94 ± 2.62% worker emergence (Fig 4 120 μL of food revealed a lower queen emergence percentage (60.07± 5.92%); however, this rate was not significantly different from that obtained when 150 μL of food was provided.However, both food levels resulted in a high dead larval percentage (Fig 4).

Microscopy analysis of the reproductive system
We compared the reproductive systems of the in vitro queens with those of naturally produced worker stingless bees to verify whether the in vitro-produced adult is a queen.Ovary size and the presence of a spermatheca were used to differentiate between queens and workers confirming the status of an emerging adult as a queen.In vitro queens from the 120 and 150 μL treatments were larger than workers (Fig 5), with a considerable abdomen and well-developed reproductive system.They possessed a large ovary with numerous ovarian filaments (ovarioles) and spermathecae (Fig 6), increasing their likelihood of laying eggs.By contrast, H. itama workers had ovaries but no spermathecae, and their ovary size was smaller than those of the in vitro queens (Fig 6).

Morphometric analysis
We assessed and compared 34 morphometric characteristics among the in vitro queens, natural virgin queens, and adult workers (S2 Table ).Discriminant analysis showed no significant differences between the in vitro and virgin queens produced under natural conditions; however, they were separated from the workers (Fig 7).Morphometric analysis demonstrated no significant difference in size between the in vitro-reared queens and natural virgin queens (p > 0.05), except for the head length (p = 0.019), antenna length (p = 0.017), length of tergite 4 (p = 0.023), and width of the upper edge of tergite 4 (p = 0.006), where the in vitro queens were found to be smaller than the natural virgin queens (S3 Table ).Furthermore, queens under both conditions were considerably larger than the workers, except for the number of mandible teeth (p > 0.05) and flagellum segments (p > 0.05) (S3 Table ).
Interestingly, workers exhibited markedly greater head width, compound eye length and width, mandible length, hind tibia length and width, hind basitarsus length, forewing length and width, marginal cell length, 1 st submarginal cell length, and hind wing length and width when compared to the in vitro and natural virgin queens (S3 Table ).

Acceptance rate of the in vitro-reared virgin queens
Over 70% of the in vitro-reared queens were accepted after being introduced to the artificial queenless colonies that consisted of young workers only (AQCa).Contrastingly, a lower acceptance rate (approximately 46%) of the in vitro-reared queens was found in the queenless colonies that consisted of young and old workers (AQCb) (Fig 8).The odds ratio (OR) test demonstrated that the in vitro-reared queens had a high acceptance rate in small artificial colonies that contained only young worker bees (OR = 2.917; 95% CI 0.594-14.).The difference in the likelihood of rejecting the in vitro-reared queens between older workers from small queenless colonies was insignificant (χ2 = 1.784, df = 1, P = 0.182).

Paternity frequency and intracolonial variation
No significant difference was found in observed mating frequency, k (t = 0.389, df = 13, P = 0.704), effective mating frequency, m e (t = 0.502, df = 13, P = 0.624), mating frequency corrected for sample size, k 9 (t = 0.810, df = 13, P = 0.442), and intracolonial relatedness, r (t = 0.295, df = 13, P = 0.772) between the natural and in vitro reared queens.Low effective paternity frequency values (2.086 ± 0.296) were observed in the H. itama queens, ranging from 1.00 to 3.37 (Table 2).Additionally, no significant difference was found in the effective paternity frequency (m e ) and the deduced paternity frequency (k 9 ) between the Nakhon Si Thamrat and Narathiwat apiaries (t = 0.108, df = 13, P = 0.916 and t = 0.371, df = 13, P = 0.716, respectively).No significant difference was observed in m e and k 9 that were used to compare the natural and in vitro reared queens within apiaries (Nakhon Si Thammarat; m e : t = 1.107, df = 6, We detected no drifting workers between or among the colonies within each apiary (Table 2).This may be attributed to the worker samples being directly collected from the brood clusters inside each colony [35,36,41].

Discussion
In this study, rearing H. itama queen larvae in vitro with 120 and 150 μL of food resulted in queens with similar body sizes to natural virgin queens after 54 days (Figs 4 and 7; S1 and S2 Tables).Larval queens reared in vitro developed for approximately 54 (54.12±1.41)days before emerging as adults in this study.This finding is consistent with that of a previous study [10], approximately 50 to 56 days and 40 and 47 days of developmental growth of the queen and worker, respectively.Previous research on in vitro stingless bee queen rearing has noted the challenge of achieving naturally sized artificial queens.Hartfelder and Engles [14] successfully produced numerous in vitro-reared queens of Scaptotrigona postica but found them smaller than naturally produced queens.Baptistella et al. [19] observed that in vitro-reared Frieseomelitta varia queens were smaller than those naturally produced.However, Menezes et al. [16] reported that adding approximately 10% more larval food than the average found in  Scaptotrigona depilis natural royal cells resulted in in vitro-reared queens with the same body size as natural queens.Queen body size is crucial because smaller queens lay fewer eggs than larger queens in some stingless bee species [42,43].
In vitro-reared queens fed with 120 and 150 μL of food exhibited larger abdomens, welldeveloped ovaries with several ovarioles, and a spermatheca, enhancing the chances of successful mating and egg laying (Figs 5 and 6).However, we observed the highest rate of queen larval mortality between the last larval stage and the beginning of the pupation phase.Similar findings were noted in Plebeia droryana, with the highest loss of in vitro-reared queen larvae between the last larval and prepupal phases [17].
Our findings revealed no significant differences in queen emergence between the two larval food levels (Fig 4).However, the reproductive organs of in vitro-reared queens fed with 120 and 150 μL larval food substantially differed from those of naturally produced workers (Fig 6).Workers had smaller ovaries with fewer ovarioles and spermathecae.These results highlight the differences in the female reproductive systems between in vitro-reared queens and workers, attributed to the different larval food quantities provided [16,20].Similarly, Razali et al. [10] reported consistent findings, demonstrating that in vitro-reared H. itama queens fed with 150 μL larval food exhibited substantially larger ovaries than natural workers.
The acceptance of in vitro-reared H. itama queens tended to be unaffected by the age of the workers.In this stingless bee species, the age of workers did not markedly impact their acceptance of in vitro-reared queens.In contrast to P. droryana, Fernando dos Santos et al. [17] found that small queenless colonies with older workers had higher rejection rates for in vitroreared queens.This study observed no effect of old workers on acceptance rates, likely because virgin queens were released using a queen cage, reducing direct contact between them and workers and minimizing the risk of old workers killing the virgin queens.In practice, young and old workers, as well as young and old brood combs should be used to establish new colonies, along with honey and pollen pots.
The mating ability of in vitro-reared queens of stingless bees was first reported by Camargo [44] who found that only one out of five in vitro-reared S. postica queens mated and successfully laid eggs.Hartfelder and Engels [14] successfully produced many in vitro-reared S. postica queens capable of mating and laying eggs.However, this study observed differences in the shapes and sizes of reproductive organs between in vitro-reared and naturally produced queens.Menezes et al. [16] demonstrated that S. depilis could mate and lay fertile eggs.Our study observed a low but not significantly different mating frequency between successfully mated in vitro-reared and naturally produced H. itama queens (Table 2).In vitro rearing of H. itama queens is an efficient method for producing numerous virgin queens with a high potential for adequate colony propagation.
In vitro queen rearing of stingless bees offers an efficient method for obtaining numerous valuable virgin queens to multiply colonies.When optimal conditions, including consistent body size, shape, reproductive organ size, and a mating frequency akin to natural queens, producing several hundred virgin queens from a selective line mother colony in a short timeframe is possible.Thus, this method holds promise from colony propagation and genetic improvement efforts [16], akin to practices in apiculture [47].Meliponiculture (stingless beekeeping), a significant economic activity, has seen a surge in honey production and crop pollination in tropical and subtropical areas.Consequently, further advancements in breeding techniques and colony management practices are imperative [16,21].

Conclusion
This study demonstrates that depositing 120 and 150 μL of larval food Heterotrigona itama royal cells led to the development of queens within 54 days.In vitro queen generation using these food quantities produced queens resembling natural queens but larger than natural workers, with well-developed ovaries and spermathecae.We observed substantial mortality, especially between the last larval stage and the start of the pupation phase.Furthermore, the age of the workers did not affect the acceptance rate of the in vitro-reared queens.Regarding mating ability, we observed a modest but non-significant decrease between successfully mated in vitro-reared queens and naturally produced queens.This study aimed to enhance colony multiplication for modern economic meliponiculture activities over a short period.This technique potentially increases the number of new colonies and the economic value for stingless beekeepers.

Fig 1 .
Fig 1. Representation of the egg-transferring phases of the in vitro rearing of the Heterotrigona itama.(A) The queen-laid egg was carefully removed from the brood combs using a sterilized beekeeping moving queen grafting tool.(B) The queen-laid egg was placed vertically on an ELISA plate and deposited in a natural brood cell.https://doi.org/10.1371/journal.pone.0306085.g001

Fig 6 .
Fig 6.Microscopy images of gonadal development in Heterotrigona itama, displaying (A) the freshly dissected reproductive system of an in vitro queen with a well-developed ovarioles structure, and (B) a dissection of female workers, highlighting significant differences in ovaries and ovariole size compared to the in vitro queen.The abbreviations are ovary (Ov), lateral oviduct (LOvD), common oviduct (COvD), and spermatheca (Spe).https://doi.org/10.1371/journal.pone.0306085.g006

Table 2 . Paternity frequency in Heterotrigona itama colonies from two commercial apiaries in Thailand.
e ), and average worker relatedness (r) are listed for each colony within each apiary.Bracketed numbers are the standard deviation (SD) and standard errors (SE).https://doi.org/10.1371/journal.pone.0306085.t002